Natural products or secondary metabolites (SMs) have been invaluable as platforms for developing front-line drugs. Between 1981 and 2006, 5% of the 1031 new chemical entities approved as drugs by the FDA were natural products and for application in cancer another 47% were natural-product-derived. In addition, SMs are major sources of innovative therapeutic agents for both bacterial and fungal infectious diseases, cancer, lipid disorders, and immunomodulation. Fungal SMs have proven to be a particularly important source of new leads with useful pharmaceutical activities. A recent literature survey of fungal metabolites, covering 1500 fungal SMs that were isolated and characterized between 1993 and 2001, showed that more than half of the molecules had antibacterial, antifungal or antitumor activity.
However, the majority of existing fungal species has not been characterized for their metabolic potential. One major roadblock in this endeavor is that some species are not culturable under laboratory conditions and/or their secondary metabolite gene clusters are silent creating manufacturing difficulties as SMs are usually complicated chemically making production via traditional synthetic routes impossible.
Previous strategies on activating fungal SMs have focused mainly on 1) activating endogenous gene clusters by either over-expressing the pathway-specific transcription factor or manipulating global regulators and 2) expressing the entire gene cluster in a heterologous host. Although successful in some cases, these strategies have significant disadvantages. As not all fungal species are amenable to genetic manipulation, strategies that focus on endogenous activation are impossible in these species. If genetic manipulations are possible, activation of an otherwise silent cluster still depends the presence of a cluster-specific transcription factor. However, not all SM clusters contain transcription factors. Another major disadvantage of overexpressing SMs is that many SMs are toxic to the host fungus, thereby making the isolation of significant amounts of the desired compound difficult.
Approaches expressing fungal gene clusters in heterologous hosts (mainly Saccharomyces cerevisiae or Aspegillus spp.) focused on amplification of the entire gene cluster including native promoters. Although these approaches led to expression of the targeted gene clusters in some cases, the use of native promoters cannot guarantee controlled activation of the genes. As a result, those clusters still remain silent in the new host in most cases. Exchange of native promoters with constitutively expressing promoters for an entire gene cluster is unfeasible up to now. Although a few constitutively promoters for fungal species are commonly used, not enough promoters are known in order to fuse all cluster genes to unique promoters. The use of the same promoter sequence for several genes of a cluster is impossible due to the yeast cloning technique applied for assembling the gene cluster in a suitable plasmid. Cloning of gene clusters is achieved by PCR-based amplification of the desired DNA region and subsequent yeast recombination-based cloning. In general, the gene cluster is amplified in several 1-2 kb pieces by use of primers with short overlapping 5′-overhangs. These fragments are co-transformed with a linearized shuttle vector into yeast cells for assembly by its recombination machinery. The yeast recombination-based system requires unique promoters be used for each gene to be expressed in the plasmid. The use of the same promoter sequences would result in incorrect assembly of the desired plasmid.
Needed in the art are improved methods and systems for producing fungal secondary metabolites.